NCR negative group 3 innate lymphoid cell (NCR−ILC3) participates in abnormal pathology of lung in cigarette smoking‐induced COPD mice

Abstract Background Natural cytotoxicity receptor negative innate lymphoid cell (NCR−ILC3) involves into mucosal homeostasis, inflammation regulation and tissue remodeling. The proportion of NCR−ILC3 is increased in the lung of smokers with chronic obstructive pulmonary disease (COPD). However, there's still few understandings on the role of NCR−ILC3 in COPD pathogenesis. Methods COPD mice were induced by cigarette smoking. The pathology in lung was detected in histology. The frequency of NCR−ILC3 (CD3‐CD45+RORγt+NkP46‐) from murine lung was detected using flow cytometry. IL‐17+RORγt+ double positive cells in lung were assessed by double immunofluorescence staining. The protein expressions of epithelial‐to‐mesenchymal transition (EMT) markers, namely E‐cadherin and Vimentin, were assessed using immunohistochemistry staining and western blotting. Results The frequency of NCR−ILC3 in lung was higher in COPD group than controls. The IL‐17+RORγt+ cells in lung from COPD mice were more than controls. E‐cadherin expression was decreased but Vimentin expression was increased in lung of COPD mice, when compared with controls. The frequency of NCR−ILC3 in lung tissues were positively correlated with mean linear intercept in lung, destructive index in lung and EMT, respectively. Conclusions NCR−ILC3 could contribute to emphysema and EMT in lung of cigarette smoking‐induced COPD, which will provide further understanding on COPD pathogenesis of immune response.


| INTRODUCTION
Innate lymphoid cell (ILC) is a heterogeneous family of the innate immune system, which is composed of three groups, namely ILC1, ILC2, and ILC3, for their phenotypes and functions. 1 Recently, ILC3s have attracted an increasing attention due to their roles in maintaining mucosal homeostasis, regulating inflammation, remodeling tissue and clearing pathogen. ILC3 is characterized by expressing retinoic acid receptor-related orphan receptor γt (RORγt) for lineage specification. Most of ILC3 co-expresses C-C motif chemokine receptor 6 (CCR6), which is the receptor for epithelial chemokine CCL20. [2][3][4][5][6] ILC3 can be further subdivided into natural cytotoxicity receptor positive (NCR + ) ILC3 and NCR negative (NCR − ) ILC3. The NCR is NKp44 in human, and is NKp46 in mice. 7,8 The NCR − ILC3 is a heterogeneous subgroup that also contains lymphoid tissue inducers (LTi) cells. 1 Interestingly, for smokers with chronic obstructive pulmonary disease (COPD), proportions of ILC subsets in lung are skewed toward NCR − ILC3 whereas non-COPD individuals have balanced proportions. 9 The NCR − ILC3 also contained LTi cells. 9 Moreover, NCR − ILC3s are enriched in lungs of severe COPD patients. 10 Thus, NCR − ILC3 may participate in the COPD pathogenesis. However, there's still few understandings on the role of NCR − ILC3 in COPD pathogenesis.
The NCR − ILC3 could produce interleukin (IL)-17 and IL-22, whereas NCR + ILC3 produce IL-22 but not IL-17. 2-6 IL-17 plays a key role in the initiation of COPD. It mediates the recruitment of inflammatory cells in the lung and is essential for fibrosis of small airway in COPD. 11 IL-17 is also required for developing emphysema in response to cigarette smoking. 12 Thus, NCR − ILC3 may contribute to COPD pathogenesis partly due to IL-17. In this study, we explored the role of NCR − ILC3 in abnormal pathology of lung in COPD.
Moreover, ILC3 was reported to activate TGF-β, which is a key mediator for tissue and mucosal repair and epithelial-to-mesenchymal transition (EMT) in airway. 13 Our previous work found that IL-17 could coordinate with cigarette to induce EMT of bronchial epithelial cells. 14 Therefore, we also investigated the effect of IL-17-produced NCR − ILC3 on EMT of lung in cigarette smoking-induced COPD mice in this study.

| Mice and cigarette smoke exposure
All mice used for experiments in this study were C57BL/6 background. Mice were maintained under pathogen-free conditions. Eight to nine-week-old male mice were exposed to CS as previously described. 15 Mice in COPD group was exposed to five cigarettes (Nanning Jiatianxia unfiltered cigarettes, 12 mg of tar and 0.9 mg of nicotine) four times every day with 30 min smoke-free intervals in a closed 0.75 m 3 room, 5 days per week for up to 20 weeks. The smoke:air ratio was 1:6. The controls were exposed to air. Each group included eight mice for administration and data analysis.

| Ethical statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of Guilin Medical University (approval number N/A), and according to the ARRIVE guideline.

| Lung function measurements
Invasive pulmonary function of mice was tested with the forced oscillation technique using the FlexiVent system (Scireq) as described previously. 16 Respiratory system elastance and compliance were captured using the Flexivent "Snapshot model." Tissue elastance was captured with a constant phase model to obtain a parametric distinction between airway and tissue mechanics.

| Histology, immunohistochemistry, and immunofluorescence staining
Mice were administrated terminal anesthesia with 2% isoflurane inhalation and then euthanized by cervical dislocation. Lungs were dissected out. One thirds of left lung inflated with fresh 4% paraformaldehyde was processed using a histological automatic tissue processor and embedded in paraffin. Three-micrometer sections were cut and respectively administrated with hematoxylin and eosin (HE) stain, Periodic Acid-Schiff (PAS) stain and Masson stain to evaluate airway inflammation, goblet cell hyperplasia and mucous secretion, and extracellular matrix in the lung tissues. 17 As previously described, 18,19 the mean linear intercept (MLI) was obtained to evaluate airspace enlargement, and the destructive index was determined to assess alveolar destruction. Briefly, the MLI was obtained by dividing the length of a line drawn across the lung section by the total number of intercepts encountered in lung. The destructive index was revealed by the percentage of destroyed air spaces.

| Western blot analysis
Two thirds of left lung were subjected to western blot analysis as described. 18 Briefly, lung tissues 40 mg were homogenized, and then centrifuged. The proteins were subjected to a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring onto PVDF membranes. The membranes were incubated with rabbit anti-mouse E-cadherin (ab76319; Abcam Inc), rabbit antimouse Vimentin (ab92547; Abcam Inc), or rabbit antimouse GAPDH (ab8245; Abcam Inc) primary antibodies at 4°C overnight, and then were incubated with HRPconjugated goat anti-rabbit (ab205718, Abcam Inc) for 2 h at room temperature. The housekeeping protein was GAPDH. Finally, the blots were developed with the ECL Plus reagents (Thermo pierce).

| Statistical analysis
Data were displayed as means ± Standard deviation (SD). Comparisons between groups were analyzed using unpaired t-test. Correlation coefficients were calculated  using Pearson's method. Results were considered statistically significant for p < .05. All statistical tests were performed using SPSS 21.0 (IBM SPSS Inc).

| Pathological injury in lung tissues
In histology of lung tissues, COPD mice had increased inflammation ( Figure 1A), goblet cell hyperplasia and mucous secretion ( Figure 1B) extracellular matrix ( Figure 1C), and smooth muscle thickening ( Figure 1D), when compared with controls. That showed chronic and pathological injury in lung from COPD.
In morphology of lung, the MLI and destructive index in COPD mice were both increased when compared with controls ( Figure 1E,F). Those showed more serious destruction of lung structure, particularly airspace with an enlargement, in COPD group than controls.
For lung function, COPD group had decreased total respiratory system elastance and tissue elastance, along with increased compliance, compared with control group (Figure 1G-I). That showed functional consequences in the model of COPD mice. 3.2 | Frequency of NCR − ILC3 was increased in lung tissues from COPD mice The NCR − ILC3 was identified as CD3-CD45+RORγt +NkP46-using flow cytometry. The frequency of NCR − ILC3 in lung tissues from COPD mice was higher than that from controls (COPD vs. controls = 52.59 ± 8.56% vs. 22.01 ± 6.45%) (Figure 2). This result showed an increase of NCR − ILC3 in lung from COPD mice.

| Expressions of NCR − ILC3 related cytokines were increased in lung tissues from COPD mice
The expressions of IL-17, RORγt, CCR6, and CCL20 in lung tissues were explored by immunohistochemistry staining. All of IL-17, RORγt, CCR6, and CCL20 were showed an increase of expression in lung tissues from COPD mice, when compared with controls ( Figure 3).
To explore the activity of NCR − ILC3 in small airway, the IL-17+RORγt+ double positive cells in small airways was investigated using immunofluorescence staining. In COPD mice, IL-17+RORγt+ cells were more than that in controls ( Figure 4). The results support that NCR − ILC3 has more activity in lung of COPD mice than controls.

| Epithelial-mesenchymal transition (EMT) was increased in lung tissues from COPD mice
The protein expressions of E-cadherin and Vimentin in lung tissues were assessed using western blotting. E-cadherin protein expression was lower in COPD mice than that in controls, whereas Vimentin was expressed higher than that in controls ( Figure 5A-C). Moreover, the immunohistochemistry staining of E-cadherin and Vimentin was showed similar results as those from western blotting ( Figure 5D). Those results suggested that COPD mice had an increased EMT in lung than controls.

| Correlations
When all mice were considered together, the frequency of NCR − ILC3 in lung tissues was positively correlated with MLI (r = .718, p = .002), destructive index in lung (r = .758, p = .001) and protein expression of Vimentin in lung tissues (r = .751, p = .001), respectively ( Figure 6A,B,D). In contrast, the frequency of NCR − ILC3 in lung tissues was negatively correlated with protein expression of E-cadherin in lung tissues (r = −.818, p < .001) ( Figure 6C).

| DISCUSSION
Our present study confirmed an increase of NCR − ILC3 in lung tissues of cigarette smoking-induced COPD mice, which were demonstrated to relate with emphysema and EMT in lung.
Our results showed that the frequency of NCR − ILC3 in lung tissues from COPD mice was higher than that from controls. And IL-17+ cell, RORγt+ cell, CCR6+ cell and IL-17+RORγt+ cell were more in lungs of COPD mice than controls, which may be related with an activation of NCR − ILC3 in response to cigarette smoking. Our findings were in consistent with previous studies on smokers with COPD patients, 9,10 and further demonstrated that cigarette smoking could induce an increase of NCR − ILC3 in lung of COPD. Thus, our results suggested that in COPD patients with cigarette smoking history, cigarette smoking may induce NCR − ILC3 in the occurrence and development of COPD.
In the present study, smoking-induced COPD mice were showed significant emphysema in lungs. The MLI and destructive index confirmed the structure destruction in lung parenchyma of COPD mice. The lung function of COPD mice also demonstrated the pathological injury and functional decrease. The frequency of NCR − ILC3 in lung tissues was positively related with lung destruction in COPD mice, suggesting that NCR − ILC3 may participate in lung destruction in COPD. Since IL-17 is crucial for emphysema development in response to cigarette smoking 12 and NCR − ILC3 is one of the important sources of IL-17, 20 NCR − ILC3 may be involved into emphysema via producing IL-17. Neutralization of IL-17 may inhibit the effect of NCR − ILC3 on lung injury in COPD.
The EMT markers, namely E-cadherin and Vimentin, were assessed in the lung of cigarette smoking-induced COPD mice in our study. Our results showed that E-cadherin protein expression was lower in COPD mice than that in controls, whereas Vimentin expression was higher than that in controls. The E-cadherin+ cell and Vimentin+ cell distributions in small airways were in consistent with the protein expressions. Those results suggested that cigarette smoking could induce EMT in the lung of COPD mice, particularly in small airways, which was in consistent with previous findings. 21 Furthermore, the EMT was positively correlated with the frequency of NCR − ILC3 in lung tissues of this present study, suggesting that NCR − ILC3 may be involved into airway EMT in COPD. That may be related with IL-17, since IL-17 could coordinate with cigarette to induce airway EMT. 14 In addition, it may be associated with other mechanism, such as TGF-β, which could be activated by ILC3. 13 The precise mechanism of NCR − ILC3 in airway EMT of COPD should be further investigated in in vitro study in future.
We identified the NCR − ILC3 using markers as CD3-CD45+RORγt+NkP46-. RORγt has been found to express in immune cells including CD4 and CD8 double positive thymocytes in thymus, 22 Th17, 23 Tc17, 24 regulatory T cells, 25,26 invariant natural killer T cells (iNKT), 27 γδ T cells, 28 NK cells 29 and ILC3s. 1 The negative CD3 markers could exclude T cells and iNKT. The negative NCR could exclude NK cells, since NCR is reliable marker for NK cells. 30 Therefore, the markers CD3-CD45+RORγt+NkP46-could identify NCR − ILC3 in our study. However, it should be aware that the NCR − ILC3 remains a heterogeneous group that also encompasses LTi cells.

| CONCLUSION
In conclusion, NCR − ILC3 could contribute to emphysema and EMT in lung of cigarette smoking-induced COPD, which will provide further understanding on COPD pathogenesis of immune response.